Polyelectrolyte complexes (PEC) are produced by the interfacial complexation of polyelectrolytes. This process is mainly driven by the electrostatic bonds formed between the two oppositely charged polyelectrolytes. One of the great advantages of this process is its compatibility with mild conditions, namely room temperature, aqueous environment and physiological pH, and its non-deleterious effect on cells or drugs. Thence, cells, drugs, small peptides and others can easily be incorporated in structures produced by polyelectrolyte complexation without compromising their viability and bioactivity. PEC shape and size is easily adjustable (e.g. capsules (3D), fibers, membranes (2D), sacs (with an aperture), and microbioreactors) making possible their use on a plethora of biomedical applications. On a tissue engineering perspective, the possibility to include different components—as cells and drugs—into very specific structures is attractive, thus composing a multicomponent system. PEC can then be used alone as scaffolds or in combination with hydrogels as a way to enhance their mechanical properties.
Gellan gum is a linear anionic heteropolysaccharide secreted by the bacteria Sphingomonas elodea. Its molecular structure is based in one repeating unit consisting of glucose-glucuronic acid-glucose-rhamnose. In the native form, or high acyl form, two types of acyl substituents are present: acetyl and L-glyceryl. Low acyl gellan gum is obtained through alkaline hydrolysis of native gellan gum, which removes both of the acyl residues. Both forms of gellan gum form hydrogels in presence of metallic ions, and upon temperature decrease.
It is acid and heat resistant and has a free carboxylic group per repeating unit, which can be used for improvement of functionalization. Moreover, the presence of this carboxylic group confers to gellan gum a negative charge at neutral pH (pKa=3.1). Thus, gellan gum can be combined with positively charged polymers in order to prepare PEC systems.
Poly-L-lysine is a lysine homopolymer. Therefore, it results from the polymerization of a lysine aminoacid. Since lysines have two different amines, polymerization process can start either from the amine located at ε-carbon (ε-polylysine) or from the amine located at the α-carbon (α-polylysine), which applies to poly-L-lysine. Due to the presence of a positively charged hydrophilic amino group, poly-L-lysine is positively charged at physiological pH.
Several patent documents are based on the application of PEC for different purposes, including cell encapsulation.
U.S. Pat. No. 5,334,640 refers to crosslinked biocompatible compositions comprising an ionically crosslinked component and a covalently crosslinked component for encapsulating biologic compounds. Crosslinkable mixtures and method are also disclosed.
Document WO 2013133705 A1 relates to a composition comprising a polyelectrolyte complex, and comprising at least one biocide. A preferred composition comprises lignosulfonate and chitosan. The document further relates to methods for generating a composition and to uses of a mixture for protecting an agricultural plant or plant part against a pathogen.
U.S. Pat. No. 9,005,662 B2 describes a polyelectrolyte complex comprising an interpenetrating network. The polyelectrolyte complex further comprises a plurality of closed-shell pores, having at least one average transverse dimension between about 100 nanometers and about 1000 micrometers.
Document WO 2000001373 A1 defines a method for encapsulating a core material within a capsule having a permeable or semipermeable membrane. A complex formation reaction between oppositely charged polymers is used wherein one of the polymers is an oligosaccharide such as chitosan and the reaction is carried out at a pH between about 6.6 and 7.5.
Document WO 2004032881 A3 relates to compositions for treating a disease by implanting encapsulated biological material into a patient. The components of the used coatings can include natural and synthetic polymers, macromers, accelerants, co-catalysts, photo-initiators, and radiation and can be manipulated on order to obtain different degrees of biocompatibility, protein diffusivity characteristics, strength, and biodegradability.
These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.
General Description
The present disclosure provides a PEC systems based on gellan gum and poly-L-lysine. These systems may be processed with different architectures, including capsules, micro- and nanofibers, membranes, sacs with inner micro-nanofibers or hydrogels, and microbioreactors, and it is useful for tissue engineering and regenerative medicine applications, as well as for drug delivery.
Taking advantage of self-assembly processes, as the aforementioned PEC, it is possible to develop stable materials with specific tailored design. As they are oppositely charged, in particular gellan gum and poly-L-lysine can be used as platform to obtain self-assembly PEC systems.
Herein, it is disclosed the use of a gellan gum-based polyelectrolyte complex that can be applied for cell encapsulation purposes. Although a number of hydrogel systems based in natural polymers (e.g., alginate and chitosan) have been developed to tackle cell encapsulation, they still present several problems such as variability of production, poor nutrient supply, insufficient mechanical properties or desired stability and tuned permeability to cells. Using the aforesaid PEC, it is possible to construct a thin membrane with adjusted permeability that surrounds a biocompatible core, preferably liquefied. This membrane allows the inflow of essential nutrients and oxygen and outflow of cell waste and metabolites while blocking the recognition by immune cells. This immunoprotective device permits cell transplantation-based therapies without the using of deleterious immunosuppression drugs.
One aspect of the present subject-matter discloses a composition for use in veterinary or in human medicine comprising a polyelectrolyte complex comprising a cationic polymer, poly-L-lysine, and a gellan gum.
In one embodiment for better results, the polyelectrolyte complex material of the present subject-matter may comprises a gellan gum and a poly-L-lysine. In particular suitable for tissue engineering and regenerative medicine applications or as drug delivery systems.
In one embodiment for better results, the composition of the present subject-matter may comprise 0.05-1% (w/v) of poly-L-lysine, 0.5-10% (w/v) of gellan gum.
In other embodiment for better results, the composition of the present subject-matter may comprise 0.05-0.1% (w/v) of poly-L-lysine, 0.5-3% (w/v) of gellan gum. Preferably 0.5-1.5% (w/v) of gellan gum; more preferably, 0.5-1.5% w/v of low acyl GG.
In other embodiment for better results the gellan gum may be a low-acyl gellan gum, a high-acyl gellan gum, a methacrylated gellan gum, and combinations thereof.
In another embodiment for better results the gellan gum acylation degree may be from no acyl groups up to two acyl substituents—acetate and glycerate—both located on the same glucose residue.
In another embodiment for better results the gellan gum acylation degree may be one glycerate per repeat and one acetate per every two repeats.
In another embodiment for better results the methacrylated gellan gum may comprises a methacrylation degree up to 10%, preferably between 0.5-5%, more preferably 1-3%, even more preferably 1-1.5%. Formulations of gellan gum with different degrees of acylation (from low to high) and poly-L-lysine serve as precursor materials. When in contact, these materials interact and form a stable polyelectrolyte complex material with tunable shape and size. The material can be then tailored to form capsules to withstand the encapsulation of human and animal cells and/or drugs; fibres; 3D structures or scaffolds; micro- or nanoparticles; and any combination thereof.
In other embodiment for better results the gellan gum molecular weight may be between 5-10×104 Da (methacrylated), 2-3×105 Da (low acyl) and 1-2×106 Da (high acyl).
In another embodiment for better results the poly-L-lysine molecular weight may be between 30-500 kDa, preferably between 70-300 kDa. By varying the molecular weight of gellan gum and poly-L-lysine it is possible to adjust both biological and physicochemical properties (such as strength, flexibility, softness, degradability, chemical resistance and permeability) of the polyelectrolyte complex material, to meet specific needs.
In another embodiment for better results the composition of the present disclosure may further comprise an anti-inflammatory agent, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, a biological cell, a biological tissue and combinations thereof. Preferably, may comprise an animal or human cell, or stem cell, or combinations thereof. More preferably, may comprise an animal or human pancreatic β-cell.
In another embodiment for better results the composition may further comprise a plurality of hydrogels. More preferably, the second, or more hydrogels, is selected from a list consisting of carbopol, Matrigel®, hyaluronic acid, carboxymethylchitosan, dextran, alginate, collagen, and mixtures thereof.
In another embodiment for better results the composition may further comprise a coupling agent, in particular the coupling agent may be selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, glutaraldehyde, epichlorohydrin, dianhydrides, diamines, and mixtures thereof.
Another aspect of the present invention relates with the use of the composition of the present subject-matter in the treatment of diseases that involve the repair or regeneration of tissues; or the use as a drug delivery system; or use in cell therapy or advanced-therapy medicinal product.
Another aspect of the present invention relates with the use of the composition of the present subject-matter in the treatment or prevention of diabetes; in particular for the treatment of type 1 diabetes mellitus, type 2 diabetes mellitus, or gestational diabetes; or obesity, ageing related-diseases, tumours or pancreatic diseases.
Another aspect of the present invention relates to the use of the composition of the present subject-matter in the treatment or prevention of female infertility, in particular the use of the sac/membranes/capsules for ovarian protection, or for ovarian follicle protection or for ovocyte protection.
Another aspect of the present invention relates to capsule, sac, fibre, membrane, or microbioreactors comprising the compositions of the present subject-matter.
In another embodiment the external size of the capsules may be less than 20 mm, in particular up to 3 mm.
In another embodiment the sacs may comprise inner fibres, or hydrogel, or combinations thereof. In particular, the inner fibres are microfibres, or nanofibres or combinations thereof.
Throughout the description and claims the word “comprise” and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the disclosure will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the disclosure. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present disclosure. Furthermore, the present disclosure covers all possible combinations of particular and preferred embodiments described herein.